酵母细胞表面展示技术及其在非水相酶催化合成中的应用

酵母展示技术是将外源蛋白与酵母细胞壁蛋白融合,并将外源蛋白表达在酵母细胞表面。酵母展示技术已广泛应用于各种功能蛋白的表达及筛选。以下重点介绍酵母展示技术在脂肪酶展示体系构建及其在脂肪酸甲酯、短链芳香酯及糖酯生物合成中的应用。     

菌体表面表达与活菌疫苗

展示表达是将目的蛋白基因与细胞表面结构蛋白融合,使目的蛋白表达并锚定于细胞表面的一项技术,微生物特别是细菌常用作展示表达的宿主。在大肠杆菌中,多种外膜蛋白、鞭毛蛋白、菌毛蛋白、脂蛋白等均已被用于表达和锚定外源蛋白,革兰氏阴性菌中,较明确的是蛋白Ａ可用以细胞表面的整合,Ｍ６蛋白也被尝试用于乳酸菌的展示表达,酵母表达体系中,将目的蛋白与凝集素融合可表达细胞表面,菌体表面表达的蛋白容易被免疫系统识别,能引起强烈的免疫反应。其中沙门菌、大肠杆菌、链球菌等展示表达的致病细菌或病毒的抗原和毒素用于免疫动物均能产生高滴度的中和抗体,用作疫苗大有前途。本文对表达体系和在疫苗方面的应用进行了概述     

酵母细胞表面展示载体的构建及功能验证

目的:构建酵母细胞表面展示载体。方法:通过酶切连接方法在pADH1载体中插入信号肽、α-凝集素(含连接子)编码序列构建表面展示载体,并用EGFP来验证该载体的功能。结果:得到了267 bp的信号肽序列、1 623 bp的凝集素编码序列和717 bp的绿色荧光蛋白编码序列,酵母细胞表面展示载体被成功构建,且绿色荧光蛋白被插入到pADH1-agg中后,阳性转化子能在荧光显微镜下呈现绿色荧光。结论:这说明酵母细胞表面展示载体已经构建成功,并能成功表达和展示蛋白在酵母细胞表面。该载体的构建成功将为利用酵母表面展示系统表达和展示相关蛋白提供了平台。     

酿酒酵母表面展示表达系统及应用

酵母细胞表面展示表达系统是一种固定化表达异源蛋白质的真核展示系统,即把异源靶蛋白基因序列与特定的载体基因序列融合后导入酵母细胞,利用酿酒酵母细胞内蛋白转运到膜表面的机制（GPI锚定）使靶蛋白定位于酵母细胞表面并进行表达。它利用细胞表面展示技术使外源蛋白固定化于细胞表面,从而生产微生物细胞表面蛋白,可应用于生物催化剂、细胞吸附剂、活疫苗、环境治理、蛋白质文库筛选、高亲和抗体、生物传感器、抗原／抗体库构建、免疫检测及亲和纯化、癌症诊断等领域。国内对这一方面研究较少,本文主要介绍了该技术的基本原理、研究现状、应用及其发展前景。     

酵母表面展示技术应用的最新进展

酵母表面展示(yeast surface display, YSD)技术是一种将外源靶蛋白基因序列与特定的载体基因序列融合后导入酵母细胞，利用酵母细胞内蛋白转运机制将靶蛋白表达并定位于酵母细胞表面的技术，最常用的是α-凝集素表达系统。酵母细胞具有真核细胞翻译后修饰机制，能够帮助目的蛋白正确折叠，可以用来展示各种真核蛋白，包括抗体、受体、酶和抗原肽等。酵母表面展示技术已成为生物技术和生物医学领域的强大蛋白质工程工具，结合流式细胞分选可用于改善蛋白质性质，包括亲和力、特异性、酶功能和稳定性等。本文从文库构建与筛选、抗体工程、蛋白质工程、酶工程和疫苗开发等方面对酵母表面展示技术应用最新进展进行了综述。     

HIV-1 gp41的酵母表面展示及表达优化

以His标签检测蛋白的表达, 利用酿酒酵母表面展示系统, 成功地将HIV-1 gp41片段锚定在酵母表面, 并检测到gp41的活性。以pMD18T-gp41为模板, 通过PCR技术克隆了gp41基因, 将gp41基因通过双酶切连接到载体pICAS-His上,构建了gp41酵母表面展示载体, 并将其转化至酿酒酵母(Saccharomyces cerevisiae)MT8-1中。重组菌经培养, 利用免疫荧光染色方法进行染色, 显微镜观察发现重组酵母细胞表面有绿色荧光, 流式细胞仪结果进一步证实gp41正确折叠展示于酵母细胞表面。采用不同浓度的葡萄糖培养基进行表达优化。当葡萄糖浓度为1%时, 82.46%的酵母细胞表达了gp41抗原; 随着葡萄糖浓度升高, 蛋白表达受到抑制。     

HIV-1 gp41的酵母表面展示及表达优化

以His标签检测蛋白的表达, 利用酿酒酵母表面展示系统, 成功地将HIV-1 gp41片段锚定在酵母表面, 并检测到gp41的活性。以pMD18T-gp41为模板, 通过PCR技术克隆了gp41基因, 将gp41基因通过双酶切连接到载体pICAS-His上,构建了gp41酵母表面展示载体, 并将其转化至酿酒酵母(Saccharomyces cerevisiae)MT8-1中。重组菌经培养, 利用免疫荧光染色方法进行染色, 显微镜观察发现重组酵母细胞表面有绿色荧光, 流式细胞仪结果进一步证实gp41正确折叠展示于酵母细胞表面。采用不同浓度的葡萄糖培养基进行表达优化。当葡萄糖浓度为1%时, 82.46%的酵母细胞表达了gp41抗原; 随着葡萄糖浓度升高, 蛋白表达受到抑制。     

酿酒酵母细胞表面展示技术在燃料乙醇生产中的应用及研究进展

酿酒酵母Saccharomyces cerevisiae细胞表面展示表达系统是一种固定化表达异源蛋白质的真核展示系统,具有糖基化作用及蛋白翻译后折叠等优势,更利于基因工程操作。近年来,酵母细胞表面工程作为一种新兴策略来固定化淀粉水解酶、纤维素水解酶以及木聚糖降解酶,从而应用于燃料乙醇的生产。文中着重介绍了酵母细胞表面展示系统的基本原理、研究现状以及在生物乙醇生产中的应用前景及所面临的挑战。     

酵母表面展示T载体构建及目的蛋白的表面展示

构建一种能对PCR产物进行直接克隆并展示于酵母表面的新型T载体。根据酵母表面展示载体p YD1多克隆位点序列设计出利用两端带有XcmⅠ内切酶酶切位点的含有黄色荧光蛋白基因的XcmⅠ酶切盒,通过NheⅠ和XhoⅠ酶切位点插入到p YD1载体上形成质粒p YD-YFP,并对其进行酶切鉴定和DNA测序分析,再经XcmⅠ酶切后形成两端带有d T的表面展示T载体。利用PCR扩增两个含有荧光蛋白的融合蛋白PCAD-CFP和PSR-Ds Red的基因并直接克隆到所构建的T载体中,检测其表达功能。酶切鉴定和DNA测序结果显示PCAD-CFP和PSR-Ds Red正确插入载体上,分别转化至酿酒酵母EBY100中,激光共聚焦显微镜下观察到相应的荧光的酵母,表明克隆有融合蛋白基因片段的载体成功在酵母细胞中进行表面展示,证明了所构建的酵母表面展示T载体具有直接克隆和表面展示目的蛋白的功能。     

酵母表面展示酶技术

酵母表面工程是利用载体蛋白将外源蛋白以活性形式锚定于酵母细胞外表面,免去了外源蛋白的纯化和固定,并且对其有稳定作用。本文综述了酵母表面展示技术的原理、步骤、优点以及目前常见的酵母表面展示酶,如淀粉水解酶、纤维素水解酶、与木糖利用相关的酶、脂肪酶、有机磷水解酶的构建及应用。     

酵母表面展示体系的构建及在纤维素降解中的应用*

纤维素是来源广泛且储量较大的低成本可再生资源,但其结构致密难以利用。目前降解纤维素需要多种纤维素酶协作,而游离纤维素酶成本高、难以重复利用等问题限制了其广泛应用。利用酵母表面展示技术,可以将多个纤维素酶分别与锚定蛋白融合后共展示在细胞表面,从而构建酵母表面展示纤维素酶体系。这一体系可高效降解纤维素,一方面可以充分发挥表面展示的优点,如易回收、稳定性好、操作简单、成本低;另一方面可以将纤维素有效地降解为葡萄糖,并具有代谢产生物乙醇的潜力。阐述了酵母表面展示体系的构建原则,总结了影响展示体系效率的因素,介绍了这一技术在降解纤维素中的应用,为构建高效酵母表面展示纤维素酶体系及其他多酶体系提供参考。     

Development of yeast molecular display systems focused on therapeutic proteins, enzymes, and foods: functional analysis of proteins and its application to bioconversion

Molecular display systems using yeast have been developed for industrial, medical, pharmaceutical, and biological studies. Although several host cells are available to construct a molecular display system, the yeast Saccharomyces cerevisiae is a well-established and convenient organism in eukaryotes. A wide variety of prokaryotic and eukaryotic proteins have been displayed on yeast cell surfaces. In addition, functional analyses and applications to bioconversion have been performed on the cell surface, and cells are conveniently engineered by molecular display systems. In this review, we focus on the yeast molecular display system with regard to therapeutic proteins, several enzymes, and food ingredients. In addition, recent patents on molecular display using yeast cell for production of those compounds, screening technology and related techniques are introduced. Development of devices for functional analysis of created and modified proteins in the yeast display system is also described.     

Directed evolution of proteins for increased stability and expression using yeast display

The expression of recombinant proteins incorporated into the cell wall of Saccharomyces cerevisiae (yeast surface display) is an important tool for protein engineering and library screening applications. In this review, we discuss the state-of-the-art yeast display techniques used for stability engineering of proteins including antibody fragments and immunoglobulin-like molecules. The paper discusses assets and drawbacks of stability engineering using the correlation between expression density on the yeast surface and thermal stability with respect to the quality control system in yeast. Additionally, strategies based on heat incubation of surface displayed protein libraries for selection of stabilized variants are reported including a recently developed method that allows stabilization of proteins of already high intrinsic thermal stability like IgG1-Fc.     

Enhancement of display efficiency in yeast display system by vector engineering and gene disruption

Vector engineering and gene disruption in host cells were attempted for the enhancement of α-agglutinin-based display of proteins on the cell surface in yeast. To evaluate the display efficiency by flow cytometric analysis, DsRed-monomer fused with FLAG-tag was displayed and immunostained as a model protein. The use of leu2-d in the expression vector resulted in the enhanced efficiency and ratio of the accessible display of proteins. Moreover, the amount of displayed proteins in SED1-disrupted cells increased particularly during the stationary growth phase. The combination of these improvements resulted in the quantitatively enhanced accessible display of DsRed-monomer on the yeast cell surface. The improved yeast display system would be useful in a wider range of its applications in biotechnology.     

Construction of a new plasmid for surface display on cells of Yarrowia lipolytica

In this study, a new surface display plasmid (pINA1317-YlCWP110) was constructed in Yarrowia lipolytica using C-terminal anchor domain of YlCWP1 from Y. lipolytica based on plasmid pINA1317, a pre-existing auto-cloning system for heterologous protein production in Y. lipolytica. When the genes encoding enhanced green fluorescent protein (EGFP) and haemolysin derived from the bacterium Vibrio harveyi were cloned into the newly constructed surface display plasmid, respectively, and expressed in cells of Y. lipolytica, we found that the target proteins were successfully displayed on the yeast cells and 100% of the yeast cell had anchoring target proteins. It was also shown that the yeast cells displaying haemolysin had haemolytic activity towards erythrocytes from flounder, indicating that the fusion protein remained functional. Therefore, the newly constructed surface display plasmid will have many applications in different fields such as in immobilized biocatalyst, bioconversion, bioremediation, live vaccine development and ultra-high-throughput screening for the identification of novel biocatalysts because it has many unique characteristics. To our knowledge, this work constitutes the first report of a surface display expression system in Y. lipolytica.     

Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains

The yeast cell surface provides space to display functional proteins. Heterologous proteins can be covalently anchored to the yeast cell wall by fusing them with the anchoring domain of glycosylphosphatidylinositol (GPI)-anchored cell wall proteins (GPI-CWPs). In the yeast cell-surface display system, the anchorage position of the target protein in the cell wall is an important factor that maximizes the capabilities of engineered yeast cells because the yeast cell wall consists of a 100- to 200-nm-thick microfibrillar array of glucan chains. However, knowledge is limited regarding the anchorage position of GPI-attached proteins in the yeast cell wall. Here, we report a comparative study on the effect of GPI-anchoring domain–heterologous protein fusions on yeast cell wall localization. GPI-anchoring domains derived from well-characterized GPI-CWPs, namely Sed1p and Sag1p, were used for the cell-surface display of heterologous proteins in the yeast Saccharomyces cerevisiae. Immunoelectron-microscopic analysis of enhanced green fluorescent protein (eGFP)-displaying cells revealed that the anchorage position of the GPI-attached protein in the cell wall could be controlled by changing the fused anchoring domain. eGFP fused with the Sed1-anchoring domain predominantly localized to the external surface of the cell wall, whereas the anchorage position of eGFP fused with the Sag1-anchoring domain was mainly inside the cell wall. We also demonstrate the application of the anchorage position control technique to improve the cellulolytic ability of cellulase-displaying yeast. The ethanol titer during the simultaneous saccharification and fermentation of hydrothermally-processed rice straw was improved by 30% after repositioning the exo- and endo-cellulases using Sed1- and Sag1-anchor domains. This novel anchorage position control strategy will enable the efficient utilization of the cell wall space in various fields of yeast cell-surface display technology.     

Prospects for the Application of Yeast Display in Biotechnology and Cell Biology (Review)

The technology of the yeast cell surface display, which appeared 20 years ago and was based on the displaying of target proteins on the cell surface via fusion to an abundant cell wall protein finds broad application in basic and applied research. The main advantage of the cell surface display on the basis of eukaryotic microorganisms—yeast—is the opportunity for correct modification of mammalian proteins. The cell surface display is an important tool for the analysis and understanding of protein function and protein–protein interactions and for the screening of novel clones from peptide and protein libraries. This technology makes it possible to obtain cells with novel abilities, such as catalytic functions and affinity binding to valuable ligands, including rare and heavy metals. It provides the chance to use yeast in biotechnology and in bioremediation and biomonitoring of the environment. The review considers the methods of obtaining a cell surface display on the basis of the yeasts Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica, the properties of anchor proteins, and the main fields of yeast display technology.     

Yeast cell-surface display—applications of molecular display

In a cell-surface engineering system established using the yeast Saccharomyces cerevisiae, novel, so-called arming yeasts are constructed that are armed with biocatalysts in the form of enzymes, functional proteins, antibodies, and combinatorial protein libraries. Among the many advantages of the system, in which proteins are genetically displayed on the cell surface, are easy reproduction of the displayed biocatalysts and easy separation of product from catalyst. As proteins and peptides of various kinds can be displayed on the yeast cell surface, the system is expected to allow the preparation of tailor-made functional proteins. With its ability to express many of the functional proteins necessary for post-translational modification and in a range of different sizes, the yeast-based molecular display system appears uniquely useful among the various display systems so far developed. Capable of conferring novel additional abilities upon living cells, cell-surface engineering heralds a new era of combinatorial bioengineering in the field of biotechnology. This mini-review describes molecular display using yeast and its various applications.     

Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency.

Efficiency of yeast cell surface display can serve as a proxy screening variable for enhanced thermal stability and soluble secretion efficiency of mutant proteins. Several single-chain T cell receptor (scTCR) single-site mutants that enable yeast surface display, along with their double and triple mutant combinations, were analyzed for soluble secretion from the yeast Saccharomyces cerevisiae. While secretion of the wild-type scTCR was not detected, each of the single, double, and triple mutants were produced in yeast supernatants, with increased expression resulting from the double and triple mutants. Soluble secretion levels were strongly correlated with the quantity of active scTCR displayed as a fusion to Aga2p on the surface of yeast. Thermal stability of the scTCR mutants correlated directly with the secreted and surface levels of scTCR, with evidence suggesting that intracellular proteolysis by the endoplasmic reticulum quality control apparatus dictates display efficiency. Thus, yeast display is a directed evolution scaffold that can be used for the identification of mutant eucaryotic proteins with significantly enhanced stability and secretion properties.